Protein digestion



April 14, 1942. G. B'. Avnes.- pm,

PROTEIN DIGEsTIoN or'iginai Filed .June 1s, 19:59

INVENTORS y/Pf:

f proteins.

Patented Apr. 4149 i942 PROTEIN DIGESTION Gilbert B. Ayres,` Stamford, and Joseph George Niedercorn,

Riverside,

Conn., assignors American Cyanamid Company, New York, N. Y., a corporation of Maine- Griginal application June 16, 1939, Serial No.

279,473. Divided and this application November 9, 1939, Serial No. 303,557

4 Claims. (01.195-6 fest a uniformly good rate of activity in alkalies The invention relates to the controlled digestion of protein materials and more particularly to the batingof hides and skins by subjecting them to the action of tryptic enzymes of Pencz'llum roqueforti.

It has been discovered that Penicillium roqueforti produces enzymes which have suflicient Cil- . solid discrete particle condition, such as bran,v

`proteolytic activity under neutral, acid and alkaline conditions to render them commercially attractivein the bating of hides and in similar processes involving the selective digestion of It is an object of the present invention to provide these enzymes in the form of a dried proteolytic culture which is suitable for' use as a bate. It is a further object to provide a method for the bating of hides and skins using these enzymes as hates, preferably in conjunction with A a deliming agent such as ammonium sulfate.

The invention includes both the production of these enzymes from Penicillum roquefort by inoculation of a suitable nutrient medium and cultivation of lthe inoculated medium and the provision of the enzymate (enzyme-i-carrier) in dried form, and also the selective digestion oi proteins and the bating of hides andskins by subjecting the materials to the action of these proteolytic enzymes.

As illustrative of the proteolytic activity in alkaline solution of these enzymes produced by Pencz'llium roquefort there is .shown in the single ligure of the accompanying drawing a curve, the ordinates of which are given in terms of standard alkali and the abs'cissae in terms of pH values. The curve was constructed from values obtained in digesting a casein substrate (sodium caseinate) with a culture of these enzymes at increasing alkalinities of the sodium caseinate solution. The undigested casein was precipitated with a standardized solution o`f sulfurie acid and sodium sulfate and the filtrate therefrom, containing the digested casein, titrated with 0.1 N-alkali. The tests were conducted by a modification of the procedure of' Volhard and Loehlein for the determination of. proteolytic activity of enzymes described in Praktikum der physiologischen Chemie, part l, pages 258-259,

by Peter Rona, 2d ed., Julius Springer, Berlin,

1931.' The curve which was plotted for a pH range of 'il-l0, slopes ofi. sharply at ilrst and then gradually from the point of neutrality until a pH of about 9.5 is reached whereupon it again slopes off sharply downward to a pH of about 10. By inspection it will be seen from this curve that the enzymes of the present invention maniat least up to the point where the alkalinity reaches a pH value of about 9.5. I

The preparation of these enzymes is as follows:

A suitable nutrient medium in thev granular or moistened with an equal weight of water, is inoculated with a culture of Pentioillium roqueiorti and the inoculated moist bran spread out in thin layers on trays. The inoculated bran is then incubated in an oven maintained at a temperature of about 30 C. rand preferably at not higher than this temperature, and at a humidity in the o ven 'such that the atmosphere therein is saturated'but does not contain suiliclent moisture to cause deposition of the same onto the bran. The inoculated branA is maintained in the oven until sporulation occurs. After incubation for the optimum period the bran culture may be thoroughly mixed with 0.2% of cresylic acid in solution, if desired, to improve the wetting out of the bran when used in solution. The .culture or enzymate is then dried at a temperature below C.,-e. g., 40 C., and may be used vas such for hating, or an ammonium salt, e. g., ammonium sulfate, may be incorporated with thet moist mass and the mixture then dried. The bran used for thel culture may or may not be sterilized before the inoculation and likewise the culture may or may not be sterilized, although sterilization of the culture or enzymate does not appear necessary at the present time.

The enzyme maybe liberated from the'dried culture by elution with dilute solutions of various salts, such vas ammonium chloride, arnrnonium sulfate, sodium chloride, sodium sulfate, etc. andthe eluted enzymes used in other fields as digestants. l

The proteolytic enzymes may be applied to the hating of hides andskins in the form of an enzymate in any manner now practiced in the art for the application of other tryptic bates. One' method lnow vin use involves washing the hides from the :dehairing step, and adding an ammonium salt, such as'ammonium sulfate, tothe water containing the hides in order to lower their pH which is generally very high due to the stronglyalkaline conditions under which dehairi ing takes place. Before adding the ammonium salt the 'hides may be treated for removing lime blast by the addition of a suitable acid, such as hydrochloric acid, to the water bath containing the hides. After the pH of the bath has been suitably adjusted, the .enzyme bate is added thereto in an amountdetermined by the enzyme unit strength of the hate, the kind of hide or skin to be hated, the extent of hating desired, the length of the hating time and the temperature of the hating' bath.

5 lbs. of hydrochloric acid thereto, the hath showing red to methyl orange. After three minutes add lime to the bath'until it is slightly pink to phenol'phthalein. To the bath then add 31/2 lbs. of .ammonium sulfate and about 1% (based on the weight .of the kips) of enzymate prepared as described above. Run the paddle for the desiredAe length of hating time with a final temperature'l therein of 85 F. After the kips have been hated cool them to 70 F.

- While the application of these enzymes has been described with-particular reference tothe hating of hides and skins their utility is not limited to this neid. On the contrary they may be used in many other processes in which a tryptic tion is intended as illustrative and not as limiting of the invention, the scope of which is defined by the following claims.

'Ihis is a division of our copending application Serial No. 279,473, illed June 16, 1939.

What we claim is:

1. A process of hating1 hides whichcomprises subjecting them to the action of enzymes present in a dried culture of the mold Pencillium roqaeforti, said enzymes lbeing characterized hy a uniformly high rate of proteolytic activity under alkaline conditions within a pH range up to about 9.5.

2. A process of hating hides under alkaline.

'conditions within a pH range up to` abcut 9.5

which comprises subjecting them to the action of enzymes of Penicillz'um roquefort in a dried enzyme of high activity is desired; such as in de.

sizing and degumming textiles,`paper sizing, tenderizing of meat, stripping of gelatin from photographic lms and plates, manufacture of peptones, chewing gum, glue, foods, drugs and biological products or in any eld where by their use a protein or a protein degradation product cany be reduced to a4 solubility.

It will he understood that the above descriplower molecular size of increased culture `of* the mold, said enzymes being characterized by a uniformly high rate of proteolytic activity under alkaline conditions within a pH range up to about 9.5.

f3. A process of hating hides which comprises subjecting them to the action of enzymes of Penicillium roqueforti in a dried culture of the mold, said enzymes being characterized by a uniformly high rate of proteolytic activity under alkaline conditions within a pH range up toabout 9.5, and in the presence of an ammonium salt.

4. A process of bating'hides which comprises subjecting them to the action of lenzymes of Penicillium roquefortiin` a dried culture of the mold, said enzymes being characterized by a uniformly high rate of proteolytic activity under alkaline conditions within `a pH range up to about 9.5, and in the presence of ammonium sulfate.

- GILBERT B'. AYRES.

JOSEPH G, NIEDERCORN. 

